Abstract Endogenous viral elements (EVEs) are genetic remnants of viruses that have integrated into host genomes millions of years ago and retained as heritable elements passed on to offspring until present-day. As a result, EVEs provide an opportunity to analyse the genomes of extinct viruses utilizing these genomic viral fossils to study evolution of viruses over large timescales. Analysis of sequences from near full-length EVEs of dependoparvoviral origin identified within three mammalian taxa, Whippomorpha (whales and hippos), Vespertilionidae (smooth-nosed bats), and Lagomorpha (rabbits, hares, and pikas), indicates that distinct ancestral dependoparvovirus species integrated into these host genomes approximately 77 to 23 million years ago. These ancestral viruses are unique relative to modern adeno-associated viruses (AAVs), and distinct from extant species of genus Dependoparvovirus. These EVE sequences show characteristics previously unseen in modern, mammalian AAVs, but instead appear more similar to the more primitive, autonomously replicating and pathogenic waterfowl dependoparvoviruses. Phylogeny reconstruction suggests that the whippomorph EVE orthologue derives from exogenous ancestors of autonomous and highly pathogenic dependoparvovirus lineages, believed to have uniquely co-evolved with waterfowl birds to present date. In contrast, ancestors of the two other mammalian orthologues (Lagomorpha and Vespertilionidae) likely shared the same lineage as all other known mammalian exogenous AAVs. Comparative in silico analysis of the EVE genomes revealed remarkable overall conservation of AAV rep and cap genes, despite millions of years of integration within the host germline. Modelling these proteins identified unexpected variety, even between orthologues, in previously defined capsid viral protein (VP) variable regions, especially in those related to the three- and fivefold symmetry axes of the capsid. Moreover, the normally well-conserved phospholipase A2 domain of the predicted minor VP1 also exhibited a high degree of sequence variance. These findings may indicate unique biological properties for these virus ‘fossils’ relative to extant dependoparvoviruses and suggest key regions to explore within capsid sequences that may confer novel properties for engineered gene therapy vectors based on paleovirology data.

中文翻译：

---

依赖细小病毒在地质时间尺度上的进化——对设计基于 AAV 的基因治疗载体的意义

摘要 内源性病毒元件 (EVE) 是数百万年前已整合到宿主基因组中并作为可遗传元件传递给后代的病毒的遗传残余物，直到今天。因此，EVE 提供了一个机会，可以利用这些基因组病毒化石来分析已灭绝病毒的基因组，从而研究病毒在大时间尺度上的进化。对在三种哺乳动物分类群 Whippomorpha（鲸鱼和河马）、Vespertilionidae（圆鼻蝙蝠）和 Lagomorpha（兔子、野兔和鼠兔）中鉴定的依赖细小病毒来源的近全长 EVE 序列的分析表明，不同的祖先依赖细小病毒物种大约在 77 到 2300 万年前整合到这些宿主基因组中。这些祖先病毒相对于现代腺相关病毒 (AAV) 是独一无二的，并且与现存的依赖细小病毒属物种不同。这些 EVE 序列显示出以前在现代哺乳动物 AAV 中未见的特征，但看起来更类似于更原始、自主复制和致病的水禽依赖细小病毒。系统发育重建表明whippomorph EVE 直系同源物来自自主和高致病性依赖细小病毒谱系的外源祖先，据信迄今为止与水禽鸟类共同进化。相比之下，其他两种哺乳动物直系同源物（Lagomorpha 和 Vespertilionidae）的祖先可能与所有其他已知的哺乳动物外源 AAV 具有相同的谱系。EVE 基因组的计算机比较分析揭示了 AAV rep 和 cap 基因的显着整体保守性，尽管在宿主种系中整合了数百万年。对这些蛋白质进行建模，在先前定义的衣壳病毒蛋白 (VP) 可变区中发现了意想不到的变化，甚至在直系同源物之间，特别是在与衣壳的三重和五重对称轴相关的那些区域中。此外，预测的次要 VP1 的正常保守的磷脂酶 A2 结构域也表现出高度的序列变异。这些发现可能表明这些病毒“化石”相对于现存的依赖细小病毒具有独特的生物学特性，并建议在衣壳序列内探索的关键区域可能赋予基于古病毒学数据的工程基因治疗载体的新特性。在先前定义的衣壳病毒蛋白 (VP) 可变区中，特别是在与衣壳的三重和五重对称轴相关的区域中。此外，预测的次要 VP1 的正常保守的磷脂酶 A2 结构域也表现出高度的序列变异。这些发现可能表明这些病毒“化石”相对于现存的依赖细小病毒具有独特的生物学特性，并建议在衣壳序列内探索的关键区域可能赋予基于古病毒学数据的工程基因治疗载体的新特性。在先前定义的衣壳病毒蛋白 (VP) 可变区中，特别是在与衣壳的三重和五重对称轴相关的区域中。此外，预测的次要 VP1 的正常保守的磷脂酶 A2 结构域也表现出高度的序列变异。这些发现可能表明这些病毒“化石”相对于现存的依赖细小病毒具有独特的生物学特性，并建议在衣壳序列内探索的关键区域可能赋予基于古病毒学数据的工程基因治疗载体的新特性。